1PDH

CRYSTAL STRUCTURE OF P-HYDROXYBENZOATE HYDROXYLASE RECONSTITUTED WITH THE MODIFIED FAD PRESENT IN ALCOHOL OXIDASE FROM METHYLOTROPHIC YEASTS: EVIDENCE FOR AN ARABINOFLAVIN

Summary for 1PDH

• Entry DOI: 10.2210/pdb1pdh/pdb
• Descriptor: P-HYDROXYBENZOATE HYDROXYLASE, ARABINO-FLAVIN-ADENINE DINUCLEOTIDE, P-HYDROXYBENZOIC ACID, ... (4 entities in total)
• Functional Keywords: oxidoreductase
• Biological source: Pseudomonas fluorescens
• Total number of polymer chains: 1
• Total formula weight: 45304.25

Authors

Schreuder, H.A.,Eppink, M.H.M.,Van Berkel, W.J.H. (deposition date: 1994-12-01, release date: 1995-03-31, Last modification date: 2024-02-14)

Primary citation

van Berkel, W.J.,Eppink, M.H.,Schreuder, H.A.
Crystal structure of p-hydroxybenzoate hydroxylase reconstituted with the modified FAD present in alcohol oxidase from methylotrophic yeasts: evidence for an arabinoflavin.
Protein Sci., 3:2245-2253, 1994

PubMed Abstract: The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was replaced by a stereochemical analog, which is spontaneously formed from natural FAD in alcohol oxidases from methylotrophic yeasts. Reconstitution of p-hydroxybenzoate hydroxylase from apoprotein and modified FAD is a rapid process complete within seconds. Crystals of the enzyme-substrate complex of modified FAD-containing p-hydroxybenzoate hydroxylase diffract to 2.1 A resolution. The crystal structure provides direct evidence for the presence of an arabityl sugar chain in the modified form of FAD. The isoalloxazine ring of the arabinoflavin adenine dinucleotide (a-FAD) is located in a cleft outside the active site as recently observed in several other p-hydroxybenzoate hydroxylase complexes. Like the native enzyme, a-FAD-containing p-hydroxybenzoate hydroxylase preferentially binds the phenolate form of the substrate (pKo = 7.2). The substrate acts as an effector highly stimulating the rate of enzyme reduction by NADPH (kred > 500 s-1). The oxidative part of the catalytic cycle of a-FAD-containing p-hydroxybenzoate hydroxylase differs from native enzyme. Partial uncoupling of hydroxylation results in the formation of about 0.3 mol of 3,4-dihydroxybenzoate and 0.7 mol of hydrogen peroxide per mol NADPH oxidized. It is proposed that flavin motion in p-hydroxybenzoate hydroxylase is important for efficient reduction and that the flavin "out" conformation is associated with the oxidase activity.

PubMed: 7756982

Experimental method

X-RAY DIFFRACTION (2.1 Å)